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Mutation analysis with random DNA identifiers ( MARDI) catalogs P ig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats.
- Source :
- Environmental & Molecular Mutagenesis; Mar2016, Vol. 57 Issue 2, p114-124, 11p
- Publication Year :
- 2016
-
Abstract
- Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a 'clone-by-clone' approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. Environ. Mol. Mutagen. 57:114-124, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 08936692
- Volume :
- 57
- Issue :
- 2
- Database :
- Complementary Index
- Journal :
- Environmental & Molecular Mutagenesis
- Publication Type :
- Academic Journal
- Accession number :
- 112925957
- Full Text :
- https://doi.org/10.1002/em.21992